Project Summary Maintenance and regeneration of tissues such as skin, liver, blood, and muscle decrease dramatically with age; however, molecular mechanisms that could serve to coordinate tissue and organismal aging have not been elucidated. Furthermore, it is not known whether the function of one tissue is more important than others in influencing healthspan and longevity. Aging results in loss of intestinal barrier function in species ranging from flies to humans; however, the increase in vulnerability to intestinal dysfunction in older individuals is not fully understood. Here we propose to develop the Drosophila melanogaster digestive tract as a model to study the relationship between the complexes that constitute the intestinal barrier and intestinal stem cell (ISC) behavior, as well as mechanisms underlying the age-related loss of intestinal barrier function. The intestinal epithelium provides a selective barrier that permits nutrient and water transport, while preventing uptake of harmful environmental toxins and microbial contamination of interstitial tissues. In addition to its barrier function, the intestinal epithelium also serves essential metabolic and innate immune functions. In mammals, loss of intestinal barrier function and increased intestinal permeability correlates with compromised integrity of cell-cell junctions, known as tight junctions (TJs). Septate junctions (SJs), the Drosophila analog of vertebrate TJs, are important for regulating paracellular flow between apical and basal epithelial surfaces. The SJ components in the Drosophila intestine have not been fully characterized, and their potential role in maintaining intestinal integrity throughout aging has not been interrogated. Our preliminary data indicate that there are age-related changes in the expression and localization of SJ components in the Drosophila posterior midgut. For example, we find alterations at tricellular junctions (TCJ), specialized structures where three/four cells converge, as well as cytoplasmic accumulation of several SJ components. Therefore, we hypothesize that loss of SJ/TCJ function with age leads to rapid changes in ISC behavior, which contribute to loss of intestinal homeostasis in older animals. We will test this directly through the following Specific Aims: 1) To characterize the role of TCJ in regulating ISC behavior and intestinal aging phenotypes 2) To assess the role of bicellular SJ in regulating ISC behavior and intestinal aging phenotypes 3) To explore the role of Neuroglian (Nrg) in the Drosophila intestine The causal relationships between increased ISC proliferation, loss of intestinal integrity, metabolic defects, increased inflammation, and dysbiosis are not known. Here we propose to investigate age-related changes in occulding junctions as a proximal event, leading to changes in ISC behavior and, ultimately, loss of tissue homeostasis. Our findings will have major implications for the treatment of gasterointestinal diseases and disorders, chronic inflammation, and cancer, particularly in older individuals.